The success of protein-based therapeutics is revolutionizing drug development. Unlike small molecule drugs, peptide and protein-based therapeutics can target with high selectivity and specificity defective protein-protein interaction involved in human disease. Despite their success, however, there are still numerous stability and delivery issues associated with their use as therapeutic agents. For example, monoclonal antibodies (one the most successful protein-based therapeutics with several blockbuster drugs on the market and many more in clinical development) can only target extracellular molecular targets due to their inability to cross biological membranes. They are also extremely expensive to produce and are not bioavailable due to their susceptibility to proteolytic degradation. These issues have led to the exploration of alternative protein scaffolds as a source for novel types of protein-based therapeutics. In response to this important challenge, we propose the use of genetically- and chemically-encoded libraries of cyclotides for selecting specific cyclotide sequences able to modulate protein-protein interactions. Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (?30 amino acids long) that share a 3 disulfide-stabilized core characterized by an unusual knotted structure. They have several characteristics that make them ideal drug development tools. To achieve this objective we propose to use protein-splicing technology developed in the Camarero lab to generate large, genetically encoded cyclotide libraries inside live bacterial cells. These cell-based libraries wil be screened using different in-cell reporters to identify bacteria encoding active cyclotide sequences. In addition, we will also develop novel strategies for the generation and rapid screening of chemically synthesized cyclotides targeted against membrane associated extracellular receptors. Selected cyclotides will be characterized and assayed in mammalian cells to test their ability to antagonize the selected intracellular or extracellular protein targes. We will also explore the cell penetrating properties of these interesting microproteins as well as ways to improve it. Finally, we also want to study their pharmacokinetic (PK) properties and explore different approaches to improve their bioavailability.